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Oxidation, Nitrosation, and Nitration of Serotonin by Nitric Oxide-Derived Nitrogen Oxides: Biological Implications in the Rat Vascular System
NITRIC OXIDE: Biology and Chemistry Vol. 1, No. 6, pp. 442–452 (1997) Article No. NO970147
Oxidation, Nitrosation, and Nitration of Serotonin by Nitric Oxide-Derived Nitrogen Oxides: Biological Implications in the Rat Vascular System Be´atrice Blanchard,* Maryvonne Dendane,* Jean-Franc¸ois Gallard,* Chantal Houe´e-Levin,† Abdelhak Karim,‡ Didier Payen,‡ Jean-Marie Launay,‡ and Claire Ducrocq*,1 *Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, F-91198 Gif sur Yvette, France; †LPCR, Universite´ Paris-sud, F-91405 Orsay, France; and ‡Hoˆpital Universitaire Lariboisie`re 2, rue Ambroise Pare´, F-75010 Paris, France
Received March 10, 1997, and in revised form June 25, 1997
Nitric oxide synthesized in biological systems from Because NO is not very reactive in an oxygen-free buffer, a significant part of serotonin (5-HT) is transformed by NO in nondeaerated phosphate buffer, at pH 7.4, into (4-serotonyl)-4-serotonin, 4-nitrososerotonin, and 4-nitroserotonin. Dimerization and above all nitrosation occur through the HNO2 reaction in the pH 4–6 range, possibly via radical mechanism involving N2O3 . 5-HT is readily a substrate for nitrosation by HNO2 or N2O3 , whereas tyrosine was described as not very reactive under the same conditions. Peroxynitrite converts 5-HT to the (4-serotonyl)-4-serotonin and to the 4-nitro derivative. In order to evaluate whether such structural modifications could modulate the biological properties of 5-HT, arterial pressure was measured after iv bolus injection of these derivatives to anesthetized rats. Injections of the 4-nitroso- and 4-nitro-5-HT resulted in first a brief hypotensive response and did not give the subsequent hypertensive and hypotensive phases observed with 5-HT. Finally, when tested on some cloned rat 5-HT receptors stably transfected into LMTK0 cells, both 4-nitroso and 4-nitro derivatives behaved as agonists and antagonists toward 5HT1B and 5-HT2B receptors, respectively. q 1997 Academic Press
1
To whom correspondence should be addressed. Fax: 33 1 69077247.
L-arginine and oxygen by NO synthases plays crucial
roles in the regulation of a range of physiological functions, such as vasodilation, neurotransmission, and cellular immunity (1). The most reactive targets for NO appear to be the reduced transition metals of proteins such as guanylate cyclase or hemoglobin, oxygen, and the superoxide anion. These reactions account for the short half-life of NO of a few seconds in vivo (2, 3). Current interest is focused on the ability of higher oxides of NO, such as N2O3 or peroxynitrite (ONOO0), to modify peptides, proteins, and other signaling molecules. These nitrogen oxides are short-lived intermediates that are more reactive than their precursor. They are formed during the course of NO oxidation to stable nitrite or during its coupling reaction with superoxide to form nitrate (4, 5). NO-derived nitrogen oxides can also nitrosylate thiols such as cysteine, or cysteinyl residues (6, 7), and oxidize ascorbate and tocopherol. Stimulated macrophages, neutrophiles, and endothelial cells have been shown to generate NO as well as superoxide anion and probably the end-derivative, peroxynitrite. It has been suggested that peroxynitrite is formed in human atherosclerosis and in chronic inflammation (8, 9). Peroxynitrite oxidizes glutathione, ascorbate, and tocopherol and nitrates tyrosine residues in proteins (5).
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We have studied the interactions of NO-related higher oxides under aerobic conditions with serotonin (5-hydroxytryptamine or 5-HT).2 This monoamine hormone plays an important role in the central and peripheral nervous system (10), particularly in regulating blood pressure and organ blood flow (11, 12). The products of the metabolism of 5-HT are of interest not only because of their possible physiological importance but also due to their pathological potentialities. Indeed, the 5-HT levels in the Alzheimer or Parkinsonian brain have been found to be significantly lower than those in age-matched controls, apparently caused by excessive oxidation of 5HT into several unidentified forms (13). A 5-HT dimer has been characterized as the major oxidation product during the respiratory burst of human mononuclear and polymorphonuclear phagocytes (14). In this report, we show that 5-HT is chemically transformed into a dimer and its nitroso and nitro derivatives by nitrogen oxides derived from NO. Particular attention has been paid to the reactivity with nitrous acid because it is the ultimate stable oxidative species derived from NO, and could perhaps be generated in acidic cell compartments (vesicles, hypoxic cells). To assess the proposed mechanism we show the ability of NO2 radical to oxidize 5-HT into its dimer as does azide radical in parallel experiments. In order to determine whether vascular NO might be directly involved in the change of vascular tone via chemical modification of 5-HT, some vascular properties of modified serotonin were investigated. 4-Nitroso and 4-nitro-5-HT administered to anesthetized rats were found to have completely different effects on arterial pressure and cardiac frequency compared to the parent 5-HT. Finally the properties of these derivatives were tested on cloned rat 5-HT receptors stably transfected into LMTK0 cells. EXPERIMENTAL PROCEDURES
Materials. 5-HT hydrochloride was purchased from Sigma, forskolin was from Calbiochem (San 2 Abbreviations used: 5-HT, 5-hydroxytryptamine; DOI, 2,5-dimethoxy-4-iodophenyl-2-aminopropane; ABTS, 2,2 *-azino-bis(3ethylbenzthiazoline-6-sulfonic acid); IP, inositol phosphates; GTI, 5-hydroxytryptamine-O-carboxymethylglycyltyrosinamide; FScA, forskolin-stimulated cAMP accumulation.
Diego, CA); [3H]ketanserin (63.3 Ci/mmol; 1 Ci Å 37 GBq), [3H]5-HT (75 Ci/mmol), and [125I]2,5-dimethoxy-4-iodophenyl-2-aminopropane ([125I]DOI, 2200 Ci/mmol) were from New England Nuclear Research Products (Boston, MA); [125I]5-hydroxytryptamine-5O-carboxymethylglycyltyrosinamide ([125I]GTI, 1860 Ci/mmol) was from Research Biochemicals (Natick, MA). All tissue culture reagents and Hanks’ balanced salt solution were purchased from GIBCO (Grand Island, NY); all other drugs and chemicals (reagent grade) were from Sigma Chemical. Potassium phosphate and sodium azide used in g-irradiation experiments were purchased from Prolabo and sodium nitrite was from Fluka. HPLC system. Aliquots of incubation medium were analyzed with a Waters HPLC system equipped with a Berthold integrator and using a Shandon (C 18 Si, 5 mm) Hypersyl column or a Waters RCM 25 1 10 compact preparative cartridge module. Columns were eluted using a gradient of 5– 45% acetonitrile with 0.05% TFA for 50 min at a flow rate of 1 or 6 ml/min. Products were spectrophotometrically detected by measuring UV absorbance at 215 nm. Molecular mass determination. This was performed on a VG Platform mass spectrometer (VG Biotech, Manchester, UK) using an electrospray ion source and a quadrupole mass analyzer with an upper mass limit of m/z 3000, calibrated using a mixture of polyethylene glycol 300 and 600. Ten-microliter samples were introduced into the source at a flow rate of 5 ml/min (carrier solvent: 50% acetonitrile, 49% water, 1% formic acid); 4000 V was applied to the capillary and 450 V was applied to the counter electrode. The sampling cone voltage was adjusted to obtain the best sensitivity and the lowest level of fragmentation. Experiments were carried out by J.-P. Le Caer (Ecole de Physique et Chimie Industrielles de la Ville de Paris). Reactions with NO. A saturated NO solution was prepared by bubbling NO gas (Air Liquide, France), that had been passed through a deaerated concentrated sodium hydroxide solution (10 molrdm03), into a deaerated phosphate buffer (0.4 molrdm03, pH 7.4), until the concentration of dissolved NO reached 0.7–1 mmolrdm03. The concentration was measured by the amount of nitrite present in oxy-
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genated aliquots measured spectrophotometrically by the Griess reaction (4), or by the absorbance of the oxidized derivative of the 2,2*-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Aliquots of NO solution were added to a 50 mmolrdm03 ABTS solution and NO concentration was measured as a function of the 660 nm and 750 nm absorbances (e Å 12,000 and 15,000 mol01rdm3rcm01, respectively). The NO-saturated solution (900 ml) was added by syringe to 100 ml of a stirred, phosphatebuffered solution of 5-HT (10 mmolrdm03). When NO gas was passed through a solution of sodium hydroxide and then gently bubbled into a solution of 5-HT (20 mmolrdm03) in nondeaerated phosphate buffer (0.4 molrdm03, pH 7.4) for 2 min at room temperature, HPLC analysis indicated that 5-HT was entirely converted into the same compounds as those obtained with HNO2 . Their identities were confirmed by electrospray MS analysis. 5,5*-Dihydroxy-4,4*-bitryptamine 2 and 4-nitroso5-hydroxytryptamine 3. Two or 5 eq of sodium nitrite was added to a solution of 10 or 40 mmolrdm03 5-HT hydrochloride in 0.2 molrdm03 acetate buffer adjusted to pH 5. The mixture, stirred a few minutes, became purplish red and was left at room temperature. The reaction was monitored by HPLC. The dimer (12 min retention time under conditions described above) and the 4-nitroso 5-HT (28 min) derivatives were produced after 4 h of incubation and were recovered by preparative HPLC and then lyophilized. 5,5*-Dihydroxy-4,4*-bitryptamine 2 analysis. Electrospray MS: m/z 351; 1H NMR 400 MHz in D2O: CH2 –CH2 (4m, 2.3, 2.5, 2.6, and 2.7 showing nonequivalent H); H-2 (s, 7.3), H-6 (d, 7.1, J6 – 7 Å 9 Hz), H-7 (d, 7.6, J6 – 7 Å 9 Hz) slightly different from the spectrum obtained in DMSOd6 (15); lmax Å 310 nm, in water at pH 2–3. Centesimal analysis showed that the dimer was a trifluoroacetate salt containing 2.5 water molecules, Mr 678. Nitroso derivative 3 analysis. Electrospray MS: m/z 206 and 189 (MH/ 0 NH2); 1H NMR 400 MHz in D2O: CH2 –CH2 (4 m, 2.5, 2.65, 2.75, and 2.85 at 274K which collapsed to 3 signals at 2.9, 3.1, and 3.2 at 300K). Loss of the signal from C4 –H observed in the spectrum of 1 is accompanied by the disappearance of long range coupling between C4 –H and
C6 –H. Each aromatic proton gave two peaks: H-6 (2d, 4.9 and 5.05, J6 – 7 Å 9 Hz), H-2 (2s, 6.55, 6.65), H-7 (2d, 6.9, 7.0, J6 – 7 Å 9 Hz). The intensities of the 4.9, 6.55, and 6.9 signals were twice those observed at 274K and almost equal to those observed at 300K. The spectrum of 3 in acidic water indicated lmax Å 520 nm (e Å 3700 mol01rdm3rcm01) and 385 nm (e Å 3400 mol01rdm3rcm01). Centesimal analysis showed that product 3 of Mr 355 was a trifluoroacetate salt containing 2 water molecules. 4-Nitro-5-hydroxytryptamine 4. Peroxynitrite was synthesized and its concentration was determined spectroscopically, according to Beckman’s method (16). It was added to 1 ml of a stirred solution of 5-HT (1 mmolrdm03) in phosphate buffer (0.4 molrdm03, pH 7.4) at 257C. HPLC analysis was performed on a 50-ml aliquot. The potential effects of nitrite, nitrate, and H2O2 , all present in peroxynitrite, were tested allowing the latter to decompose in the phosphate buffer before the addition of 5-HT. Less than 2% of the dimer 2 formed by the peroxynitrite reaction, was produced by residual H2O2 . The prepared nitro derivative 4, purified by HPLC, lyophilized, and dried (10% yield using 3 HOONO per 5-HT) was characterized by a 420-nm absorbance in water, pH 3. The electrospray MS spectrum showed a peak at m/z 222. 1H NMR 400-MHz spectra performed in D2O showed the CH2 (t, 3.2, 4H) and the aromatic protons H-2 (s, 7.5), H-6 (d, 6.9, J Å 9 Hz) and H-7 (d, 7.7, J Å 9 Hz). The loss of the C4–H signals and the disappearance of long-range coupling between C4–H and C6–H compared to the spectrum of 1 showed that the nitro group substitution had occurred at the 4-C position. Centesimal analysis showed that it was a trifluoroacetate salt of 4-nitro 5-HT 4, Mr 392. Nitrosation kinetics of 5-HT. These were obtained by following 4-nitroso-5-HT 3 formation at 385 nm. All experiments were carried out at room temperature, using a 941 Kontron UV-visible spectrometer. Reaction with NO2 . g-radiolysis experiments were performed with a 137Cs irradiator (Cis-Bio) located at Institut Curie-section de Recherche (Orsay, France). The dosimetry was performed using Fricke’s procedure (17). Before irradiation, solutions of sodium nitrite (1002 molrdm03) or sodium azide
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SEROTONIN TRANSFORMATIONS BY NO-DERIVED NITROGEN OXIDES
(1002 molrdm03) in potassium phosphate buffer (10 mmolrdm03, pH 7.4) were equilibrated with N2O by flushing at least 60 min under permanent vortexing, thus avoiding bubbling in the solution. The NO2 radicals were created by scavenging OH radicals by nitrite ions under N2O (18), following the reaction sequence:
a transducer system (Propac; Abbott Lab, Chicago, IL). The transducer was connected to an amplifier (CGR Thomson, Les Ulis, France) linked to a digital/ analog converter (BIOPAC Mac Pac System) and a microcomputer. After a stabilization period, 5-HT, 4-nitroso-5-HT 3, or 4-nitro-5-HT 4 diluted in a constant volume of sterile water was injected into the right jugular vein. Injections were performed in random order and a 1-h delay separated each injection. Such continuous recording of arterial pressure allowed verification of the time-related peak variations in pressure and heart rate. Receptors binding assays and measurement of 5HT1B and 5-HT2B receptor responses. Stable transfections of LMTK0 cells with the rat 5-HT1B , 5-HT2A , 5-HT2B , and 5-HT3 genes, cultures of these cell lines, membrane preparation, receptor binding assays, and measurements of receptor responses, i.e., inositol phosphates (IP) accumulation (5-HT2A , 5-HT2B) or inhibition of forskolin-stimulated cAMP accumulation (FScA) (5-HT1B), were performed as described previously (19, 20).
0 / N2O r •OH / HO0 / N2 eaq •
OH / NO20 r HO0 / •NO2
The N3 radicals were created by scavenging the OH radical formed by irradiation of azide-containing solutions under a N2O atmosphere according to the following reaction: •
OH / N30 r HO0 / •N3
The yields of NO2 or of N3 radicals are thus both equal to GOH / Ge-aq , i.e., to 0.55 mmolrJ01. At a dose rate of 3.89 Gyrmin01, the NO2 radicals were created at a rate of 2.14 mmolrdm03rmin01. Hemodynamic responses in anesthetized rats. After ip injection of 50 mgrkg01 pentobarbital, the anesthetized males CD rats (380–400 g) were mechanically ventilated via tracheotomy (3 ml tidal volume, 65–70 breath rate/min). The adequacy of the ventilation was checked by serial blood gas measurements. The arterial pressure was continuously monitored in the carotid vessel after catheterisation using
H
RESULTS
When 5-HT 1 was treated at room temperature with a solution of NO in an aerobic buffered solution at pH 7.4, or by using nitrite at pH 5, it was essentially converted, in both cases, into a mixture of the symmetrical dimer 5,5*-dihydroxy-4,4*-bitryptamine 2 and of the 4-nitroso derivative 3.
O N
N
NO¤ NH¤
HO NH¤
OH HO
NH¤
HO
NH¤
N
N
N
H
H
H 2
4
3
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treated with 5 eq of nitrous acid at pH 6 was nitrosated at a rate of 3 1 1005 molrdm03rmin01, that is, tenfold slower than at pH 5 and a thousandfold slower than at pH 3. Independently of pH, the nitrosation rate was a function of the 5-HT and nitrite concentrations. The nitrosation rate was proportional to the initial nitrite concentration when the nitrite/ 5-HT ratio was higher than two (results not shown) and otherwise varied with the square of the 5-HT concentration (Fig. 2). The reaction rate was second order with respect to the concentration of 5-HT due to a complex effect of the pH on this reaction, in a similar way to 2-naphthol nitrosation (21). FIG. 1. Kinetics of dimer (h) 2 and 4-nitroso-5-HT (m) 3 formation from 5-HT (1 mmolrdm03) and nitrite (2 mmolrdm03) in acetate buffer at pH 5. Under these conditions, 5-HT decreased linearly in the 2 h first time. Concentrations were evaluated by calibrating HPLC integration with known concentrations of each compound.
The structures of 2 and 3 were established by MS and NMR studies. The 1H NMR spectrum of 3 showed the presence of two distinct stable conformers of 4-nitroso 5-HT. These structures result from a hydrogen bond between the nitroso group and the primary amine, corresponding to a fixed side chain, or a hydrogen bond involving the aromatic hydroxyl group. Increasing the temperature promoted formation of the latter conformer. Reactions of 5-HT with NO. There was a partial transformation with a single bolus of NO injected into a neutral solution of 5-HT (NO/5-HT Å 1/1 mmolrdm03), leading to 8% nitrosation and 2% dimerization. Because NO was unreactive in oxygenfree buffer, molecular oxygen is needed for NO reactivity: nitrosating agents, such as NO2 or N2O3 , may be involved in these reactions. Nitrogen oxide intermediates of NO oxidation were either rapidly transformed into nitrite or irreversibly trapped by the substrate. Furthermore, NO behaved also as a nitrating agent in the presence of oxygen as pointed out by the formation of a small quantity of 4-nitro5-HT 4 (1.2%), which was clearly not detectable in the HNO2 reaction. Reactions of 5-HT with HNO2 . The reactions with nitrous acid accounted for the pH-dependent total conversion of 5-HT to both the nitroso derivative 3 and the dimer 2 (Fig. 1). Thus, 5-HT (20 mmolrdm03)
Reactions of 5-HT with peroxynitrous acid. 5-HT was converted by peroxynitrite in aqueous buffered solutions at pH 7.4 into the nitrocompound 4 and the dimer 2. The best peroxynitrite to 5-HT ratio for obtaining the dimer was 2/1, while it was 7/1 for the nitro compound (Fig. 3). The products formed from 5-HT treated with higher excess of peroxynitrite were not detected under our conditions. We evaluated the effects of some natural antioxidants on the yields of dimerization and nitration with peroxynitrite (Fig. 4). Ascorbate and cysteine decreased the yields of products in all the reactions involved, providing 100% inhibition with an equimolar quantity of ascorbate (compared to 5-HT concentration) and 50% inhibition with cysteine. Conversely, ferric ions significantly increased (20%) the
FIG. 2. Influence of the initial 5-HT concentration on the nitrosation rate of 5-HT by nitrite (5 eq) in acetate buffer at pH 5. Nitrosation rates were evaluated by spectrophotometry using the characteristic 385-nm absorbance of the 4-nitroso-5-HT 3.
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SEROTONIN TRANSFORMATIONS BY NO-DERIVED NITROGEN OXIDES
FIG. 3. Yields of the dimer (h) 2 and the nitro derivative (j) 4 from the reaction of 5-HT (1 mmolrdm03 in phosphate buffer at pH 7.4) with peroxynitrite. 5-HT was converted linearly with peroxynitrite concentration until 3 eq. Concentrations were evaluated by calibrating HPLC integration with known concentrations of each compound.
nitration yield without affecting that of dimerization. Reaction of 5-HT with •NO2 . Table I gives the yields of compounds obtained by reaction of 5-HT with either •NO2 or azide free radicals in bufferedphosphate solutions. The amount of dimer 2 produced is almost the same for both oxidants, showing a very similar oxidation proceeding by a simple outer-sphere mechanism. As observed for tyrosine, only a part of the free radicals undergoes dimerization during the oxidation process (22). The kinetic scheme proposed is:
447
ing moderate hypotention (023%) occurring at 6 { 2 s followed by a large and significant increase in pressure (/59 { 8%; õ0.001) peaking at the 25th second after bolus injection. This hypertension was followed by a stable hypotension (037 { 6%; õ0.01). The heart rate had a different evolution: after a significant bradycardia (042.4 { 23%; õ0.001) occurring at the 6th second, the heart rate normalized thereafter. In terms of pressure response, bolus injections of the 4nitroso or 4-nitro derivatives induced initial hypotension similar to 5-HT but with no delayed hypertension (õ0.001) and a more rapid and complete pressure recovery (Fig. 6). In addition, the initial bradycardia induced by 5-HT did not occur when 5-HT was substituted on the C4 position (Fig. 7). Receptors binding assays and measurement of 5HT1B and 5-HT2B receptor responses. Since the 5HT triphasic hemodynamic pattern is attributed to the successive activation of 5-HT3-, 5-HT2-, and 5HT1-like receptors (12), the 4-substituted derivatives of 5-HT were tested on stably transfected cells with subtypes of these 5-HT receptors present in rat vasculature (23). Table II shows that both 4-nitroso-5HT 3 and 4-nitro-5-HT 4 are able to interact significantly with rat 5-HT1B and 5-HT2B receptors but not with rat 5-HT2A or 5-HT3 receptors. Both derivatives exhibit similar binding affinities which, compared to 5-HT itself, are lower for the 5-HT1B receptor and alike for the 5-HT2B one. Interestingly enough, 3 and 4 inhibit FScA (5-HT1B receptor) or DOI-induced IP
5-HT / •N3 or •NO2 r •5-HT / N30 or NO20 [a] 2(•5-HT) r 2
[b]
5-HT / NO2 r 4
[c]
•
•
Dimerization [b] should compete with the nitration [c] in the presence of •NO2 . Hemodynamic responses in anesthetized rats. Before each injection, arterial pressure did not differ from baseline. After an iv bolus injection of 5-HT, three successive phases were observed: a short-last-
FIG. 4. Influence of some factors on the yields of dimerization and nitration of 5-HT (1 mmolrdm03) by peroxynitrite (2 mmolrdm03). Concentrations were evaluated by calibrating HPLC integration with known concentrations of each compound.
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BLANCHARD ET AL. TABLE I
Yields of 5-HT Derivatives after g-Irradiation of Neutral Nitrite or Azide Solutions •
•
NO2
N3
Irradiation dose (Gy)
19.4
38.9
58.35
77.8
116.7
155.6
5-HT dimer 2 (%) Residual 5-HT (%) 4-Nitro-5-HT 4 (%)
8{1 83 { 4 10 { 1
8 {2 81 { 4 10.5 { 1
7 {1 68.5 { 2 12 { 1
8{1 64 { 3 13 { 1
8.5 { 2 54 { 2 14 { 1
13 { 2 62 { 2 0
Note. The NO2 or N3 radicals were created by scavenging OH by nitrite (1002 mol rdm03) or azide (1002 molrdm03) in phosphate buffer (1002 molrdm03, pH 7.4) under N2O. The initial concentration of 5-HT was 1004 molrdm03. Concentrations of the 5-HT derivatives were evaluated by calibrating HPLC integration with known concentrations of each compound.
accumulation (5-HT2B receptor), indicating that these derivatives behave as agonists and antagonists toward the rat 5-HT1B and 5-HT2B receptors, respectively.
There are both ionic and radical routes which could lead to formation of dimer 2 and nitroso derivative 3. Activation of the C4 position toward nucleophilic attack results from a carbocation intermediate which should give 4,5-dihydroxytryptamine in water and thus the corresponding 4,5-dione (26). A radical mechanism could be also proposed to account for both dimerization and nitrosation of 5-HT by N2O3 (27). It has been suggested that NO2 can capture a hydrogen atom from the hydroxyl group of 5-HT forming a phenoxy radical (28) which can be delocalized on the aromatic ring. This reaction occurs between 5-HT and NO2 obtained by g-irradiation of nitrite ions under N2O. In the absence of NO, dimerization of 5-HT occurs with the same rate and with the same yield whether with azide or NO2 radicals (Table I). The mechanism is close to that of one electron oxidation of tyrosine in aqueous solution and in proteins which leads to dityrosine and also to hydroxylated compounds (22, 29). The radiolytic yield of dityrosine formation using azide as oxidant is
DISCUSSION
The aim of our work was to study the reactivity of 5-HT with the oxidized derivatives of the NO radical, these entities being able to occur in mammals. Nitrous acid, the end-product of NO autoxidation as well as NO in the presence of oxygen at neutral pH, gave 4-nitroso-5-HT 3 and 5,5*-dihydroxy-4,4*-bitryptamine 2 in similar proportions (4/1, respectively). These results suggest that the same reagent, N2O3 (or the •NO2 and •NO couple), may be formed either by autoxidation of NO or through HNO2 dismutation (24, 25). All NO derivatives are oxidizing agents since they all led to dimerization of 5-HT, the major reaction product of 5-HT oxidation, as determined conclusively by electrochemical methods (15).
TABLE II
Affinity Values of 5-HT, 4-Nitroso-5-HT, and 4-Nitro-5-HT for Cloned Rat 5-HT Receptors Stably Transfected into LMTK0 Cells
5-HT 4-Nitroso-5-HT 3 4-Nitro-5-HT 4
5-HT1B
5-HT2A
5-HT2B
5-HT3
7.60 { 0.13 6.97 { 0.15 6.83 { 0.22
5.51 { 0.17 õ5 õ5
7.59 { 0.15 7.66 { 0.17 7.31 { 0.18
6.89 { 0.08 õ5 õ5
Note. Affinity values (pKD Å 0log molrdm03) were determined using the following radioligands: [125I]GTI for 5-HT1B , [3H]ketanserin for 5-HT2A , [125I]DOI for 5-HT2B , and [3H]5-HT for 5-HT3 . Nonspecific bindings were determined in the presence of 10 mM mianserin (5-HT2 sites) or 10 mM 5-HT (5-HT1B and 5-HT3 sites). Each value is the mean { SE of at least three independent trial runs in triplicate. Eight different concentrations of each competing drug were used. Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.
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449
FIG. 5. Proposed scheme for the reactivity of nitrous acid at pH 5 or NO in the presence of oxygen at pH 7.4.
around 0.1 mmol J01 at pH 7 (30), lower that of the dimer obtained from 5-HT. The dimerization is more rapid than the formation of the nitro derivative 4 with NO2 . In the presence of NO, the serotonyl radical might react more rapidly with NO than with NO2 , asserting the proposed mechanism of nitrosation and oxidation of 5-HT with HNO2 or N2O3 (Fig. 5). Nitrogen oxides derived from NO under aerobic conditions are mainly nitrosating agents and to a lesser extent nitrating agents, suggesting that NO2 is an intermediate formed in addition to N2O3 during NO autoxidation (31). In addition to oxidation, nitration is an essential reaction of 5-HT in the presence of peroxynitrite. The yield of peroxynitrite-mediated nitration increased in the presence of ferric ions, indicating an ionic mechanism involving NO2/ from peroxynitrous acid HOONO (5, 32). Dimerization and perhaps a part of nitration by peroxynitrite could also take place via a radical mechanism, in which peroxynitrous acid undergoes homolytic rupture with the subsequent formation of •OH and •NO2 . Both radi-
cals can generate a serotonyl radical from the substrate. However, the •OH-generating system (Fe2/, ascorbate, H2O2) is known to oxidize 5-HT to compounds that we have not detected: 5,6-dihydroxytryptamine, 5-hydroxy-3-ethylamino-2-oxindole, and a dimer formed by linkage between 7-(tryptamine-4,5dione)-yle and 3-(5-hydroxy-tryptamine-2-one)-yle (33, 34). The biological properties of the proposed new metabolites 3 and 4 were evaluated on arterial pressure in rats. The triphasic vascular response characteristic of 5-HT consists of an initial quick fall in arterial pressure followed by a pressor response of 40 s and finally a long-lasting depressor phase (Fig. 6). This hemodynamic pattern is admittedly related to the successive activation of 5-HT3 , 5-HT2 , and 5-HT1 receptors (12). Administration of the 4-nitroso 3 or 4nitro 4 derivatives gave the same hypotensive responses as 5-HT, corresponding to 5-HT3 and 5-HT1 agonist properties (Fig. 6). The hypertension peak (Fig. 6) observed after 5-HT administration was clearly suppressed in the case of both 4-substituted derivatives. Accordingly, both derivatives behave as
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sponses similar to those of 5-HT (Fig. 6). The longlasting depressor phase fits well with their 5-HT1B agonist properties (Table I), but the rapid initial drop of arterial pressure, attributed to 5-HT3 receptors (12), appears at variance with the apparent nonrecognition of this 5-HT receptor type by both derivatives 3 and 4 (Table I). This might be due to the cloning and expression of the only ligand binding subunit of the 5-HT3 receptor, therefore not reflecting the heterogeneity observed with this receptor, not only between but also within species (35). This might also be due to involvement of other 5-HT receptors with similar antagonist pharmacophores, such as 5-HT4 (35). This latter possibility is favored by the inability of both derivatives 3 and 4 to reproduce the 5-HT-induced bradycardia (the so-called Bezold–Jarisch reflex, Fig. 7), also attributed to 5HT3 receptors despite the multiplicity of 5-HT receptors in the heart (36). Clearly, the delineation of the pharmacological profiles of derivatives 3 and 4 deserves further investigations. CONCLUSION
The conversions of 5-HT we have observed are very similar to those described for tyrosine (32), even
FIG. 6. Typical recordings of the arterial pressure response provoked by iv bolus of 5-HT or 4-nitroso-5-HT. Mean arterial pressure obtained after 50 mg iv injection of 5-HT (black column), n Å 6, 4-nitroso-5-HT (open column), n Å 8, or 4-nitro-5HT (hatched column), n Å 5, in anesthetized rats. The arterial pressure was evaluated at time corresponding to each phase of the vascular response following 5-HT administration. Means / SE from n experiments.
antagonists toward rat 5-HT2B receptors, suggesting that this 5-HT2 subtype, and not the 5-HT2A receptor (Table I), accounts, at least partly, for these suppressive effects. Administration of the 4-nitroso 3 or 4-nitro 4 derivatives resulted in hypotensive re-
FIG. 7. Heart rate (%) in each phase following iv bolus doses of 50 mg of 5-HT (black column), 4-nitroso-5-HT (open column), or 4-nitro-5-HT (hatched column) in anesthetized rats. The mean heart rate was evaluated at time corresponding to each phase of the vascular response following 5-HT administration.
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though 5-HT is more sensitive to oxidation than tyrosine. Indeed, 5-HT has been described as a true radical scavenger of oxygen and its reduced derivatives. It also behaves as a scavenger of oxidizing and nitrating agents derived from NO. The concentration of free 5-HT in the plasma is in the picomolar range. 5-HT can be released in large amounts from its storage centers in platelets or thrombocytes and plays a protective role at sites of endothelial damage during the course of inflammation or infection. Some authors have pointed out its ability to suppress the respiratory burst and phagocytic activity and to protect endothelial cells at inflammation sites, via the dimerization of 5-HT and subsequent reduction of the reactive oxygen species (14). On the other hand, phagocytic function should be preserved because phagocytes can actively take up and store 5-HT in granules. In any interpretation of the various effects of modulation of the immune system by 5-HT, direct interactions between reactive agents derived from O2 or/and NO should be seriously considered. Numerous biological interactions between NO and 5-HT are postulated to involve recognition of 5-HT receptors and further metabolic pathways, among which are activation of NO synthase (37, 38). In the nervous and peripheral systems, the activity of 5HT (vasopressure and intestinal motility, mood and behavior) is known to be linked to the capacity of this amine to interact with its specific receptors. Chemical interactions between NO and 5-HT should modulate the biological actions of 5-HT in NO-synthase-containing cells or in tissues (39). Particular interest should be focused on behavioral abnormalities displayed in male mice lacking neuronal nitric oxide synthase, similar to those obtained with targeted disruption of 5-HT1B receptor genes or with drugs inducing 5-HT depletion (40). The detection of such irreversibly modified derivatives of 5-HT in cells or in tissues is under investigation. ACKNOWLEDGMENTS We thank Dr. Yann Henry for his valuable advice and Dr. Pierre Potier for encouragement. Financial support was from the Centre National de la Recherche Scientifique and Ministe`re de la Recherche et de la Technologie (France).
REFERENCES 1. Nathan, C., and Xie, Q. W. (1994). Nitric oxide synthases: Roles, tolls and controls. Cell 78, 915–918.
2. Ignarro, J. L. (1990). Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu. Rev. Pharmacol. Toxicol. 30, 535–560. 3. Czapski, G., and Goldstein, S. (1995). The role of the reactions of NO with superoxide and oxygen in biological systems: A kinetics approach. Free Radical Biol. Med. 19, 785–794. 4. Wink, D. A., Darbyshire, J. F., Nims, R. W., Saavedra, J. E., and Ford, P. C. (1993). Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: Determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O2 reaction. Chem. Res. Toxicol. 6, 23– 27. 5. Pryor, W. A., and Squadrito, G. L. (1995). The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268, L7699–L7622. 6. Goldstein, S., and Czapski, G. (1996). Mechanism of the nitrosation of thiols and amines by oxygenated NO solutions: The nature of the nitrosating intermediates. J. Am. Chem. Soc. 118, 3419–3425. 7. Kharitonov, V. G., Sundquist, A. R., and Sharma, V. S. (1995). Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J. Biol. Chem. 270, 28158–28164. 8. Haddad, I. Y., Pataki, G., Hu, P., Beckman, J. S., and Matalon, S. (1994). Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J. Clin. Invest. 94, 2407–2413. 9. White, C. R., Brock, T. A., Chang, L.-Y., Crapo, J., Briscoe, P., Ku, D., Bradley, W. A., Gianturco, S. H., Gore, J., Freeman, B. A., and Tarpey, M. M. (1994). Superoxide and peroxynitrite in atherosclerosis. Proc. Natl. Acad. Sci. USA 91, 1044–1048. 10. Saito, H., and Yoshioka, M. (1994). Role of serotonin receptors in the peripheral nervous system. Biogenic Amines 10, 181– 194. 11. Page, I. H., and Mc Cubbin, J. M. (1953). The variable arterial pressure response to serotonin in laboratory animals and man. Circ. Res. 1, 354–362. 12. Kalkman, H. O., Engel, G., and Hoyer, D. (1984). Three distinct subtypes of serotonergic receptors mediate the triphasic blood pressure response to serotonin in rats. J. Hypertension 2(Suppl. 3), 143–145. 13. Volicer, L., Langlais, P., Matson, W., Mark, L., and Gamache, P. (1985). Serotoninergic system in dementia of the Alzheimer type abnormal forms of 5-hydroxytryptophan and serotonin in cerebrospinal fluid. Arch. Neurol. 42, 1158–1161. 14. Schuff-Werner, P., Splettsto¨sser, W., Schmidt, F., and Huether, G. (1995). Serotonin acts as a radical scavenger and is oxidized to a dimer during the respiratory burst of human mononuclear and polymorphonuclear phagocytes. Eur. J. Clin. Invest. 25, 477–484. 15. Wrona, M. Z., and Dryhurst, G. (1987). Oxidation chemistry of 5-hydroxytryptamine. 1. Mechanism and products formed at micromolar concentrations. J. Org. Chem. 52, 2817–2825. 16. Beckman, J. S., Chen, J., Ischiropoulos, H., and Crow, J. P.
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(1994). Oxygen radicals in biological systems. Methods Enzymol. 233, 229–240. 17. Spinks, J. W. T., and Woods, R. J. (1990). Introduction to Radiation Chemistry, 3rd ed. Wiley Interscience, New York. 18. Neta, P., Huie, R., and Ross, A. B. (1988). Rate constants for reactions of inorganic radicals in aqueous solution. J. Phys. Chem. Ref. Data. 17, 1027–1284. 19. Loric, S., Maroteaux, L., Kellermann, O., and Launay, J.-M. (1995). Functional serotonin-2B receptors are expressed by a teratocarcinoma-derived cell line during serotoninergic differentiation. Mol. Pharmacol. 47, 458–466. 20. Kellermann, O., Loric, S., Maroteaux, L., and Launay, J.-M. (1996). Sequential onset of three 5-HT receptors during the 5-hydroxytryptaminergic differentiation of the murine 1C11 cell line. Br. J. Pharmacol. 118, 1161–1170. 21. Castro, A., Iglesias, E., Leis, J. R., Mosquera, M., and Pena, E. (1987). Kinetics of the C-nitrosation of 2-naphthol and its catalysis by nucleophiles and buffers. Bull. Soc. Chim. 83– 88. 22. Garrison, W. M. (1987). Reaction mechanisms in the radiolysis of peptides, polypeptides, and proteins. Chem. Rev. 381– 398. 23. Ullmer, C., Schmuck, K., Kalkman, H. O., and Lu¨bbert, H. (1995). Expression of serotonin receptor mRNA in blood vessels. FEBS Lett. 370, 215–221. 24. Kurosky, A., and Hofman, T. (1972). Kinetics of the reaction of nitrous acid with model compounds and proteins, and the conformational state of N-terminal groups in the chymotrypsin family. Can. J. Biochem. 50, 1282–1296. 25. Beake, B. D., Moodie, R. B., and Sandall, J. P. B. (1994). The kinetics and mechanism of oxidation of hydroquinone and chloroquinone in the presence of nitrous acid in aqueous acid solution. J. Chem. Soc. Perkin Trans. 957–960. 26. Wrona, M. Z., and Dryhurst, G. (1990). Electrochemical oxidation of 5-hydroxytryptamine in aqueous solution at physiological pH. Bioorg. Chem. 18, 291–317. 27. de la Brete`che, M.-L., Billion, M.-A., and Ducrocq, C. (1996). Reactivity of nitrite with 2-chlorophenol, t-butyl phenol and resorcinol in mild acidic conditions. Bull. Soc. Chim. Fr. 133, 973–977. 28. Park, J. Y., and Lee, Y. N. (1988). Solubility and decomposition kinetics of nitrous acid in aqueous solution. J. Phys. Chem. 92, 6294–6302.
29. Hudggins, T., Well-Knecht, M. C., Detories, N. A., Baynes, J. W., and Thorpe, S. R. (1993). Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation. J. Biol. Chem. 268, 12341–12347. 30. Najari, H., Lmoumene-El Hanine, C., Faraggi, M., and Houe´e-Levin, C. (1997). Influence of N-acetyl cysteine on the one electron oxidation of tyrosine containing peptides. To be published. 31. Goldstein, S., and Czapski, G. (1995). Kinetics of nitric oxide autoxidation in aqueous solution in the absence and presence of various reductants. The nature of the oxidizing intermediates. J. Am. Chem. Soc. 117, 12078–12084. 32. Van der Vliet, A., Eiserich, J. P., O’Neill, C. A., Halliwell, B., and Cross, C. E. (1995). Tyrosine modification by reactive nitrogen species: A closer look. Arch. Biochem. Biophys. 319, 341–349. 33. Wrona, M. Z., Yang, Z., McAdams, M., O’Connor-Coates, S., and Dryhurst, G. (1995). Hydroxyl radical-mediated oxidation of serotonin: Potential insights into the neurotoxicity of methamphetamine. J. Neurochem. 64, 1390–1400. 34. Wrona, M. Z., Goyal, R. N., Turk, D. J., LeRoy Blank, C., and Dryhurst, G. (1992). 5,5*-Dihydroxy-4,4*-bitryptamine: A potentially aberrant, neurotoxic metabolite of serotonin. J. Neurochem. 59, 1392–1398. 35. Gaster, L. M., and King, F. D. (1997). Serotonin 5-HT3 and 5-HT4 receptor antagonists. Med. Res. Rev. 17, 163–214. 36. Saxena, P. R., and Villalon, M. C. (1991). 5-Hydroxytryptamine: A chameleon in the heart. Trends Pharmacol. Sci. 12, 223–227. 37. Wakabayashi, I. (1993). Involvement of nitric oxide in fading of 5-hydroxytryptamine-induced vasoconstriction. Pharmacol. Toxicol. 72, 175–181. 38. Fozard, J. R. (1995). The 5-hydroxytryptamine–nitric oxide connection: the key link in the initiation of migraine? Arch. Int. Pharmacol. Ther. 329, 111–119. 39. Leonard, C. S., Kerman, I., Blaha, G., Taveras, E., and Taylor, B. (1995). Interdigitation of nitric oxide synthase-, tyrosine hydroxylase-, and serotonin-containing neurons in and around the laterodorsal and pedunculopontine tegmental nuclei of the guinea pig. J. Comp. Neurol. 362, 411–432. 40. Nelson, R. J., Demas, G. E., Huang, P. L., Fishman, M. C., Dawson, V. L., Dawson, T. M., and Snyder, S. H. (1995). Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase. Nature 378, 383–386.
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Oxidation, Nitrosation, and Nitration of Serotonin by Nitric Oxide-Derived Nitrogen Oxides: Biological Implications in the Rat Vascular System Be´atrice Blanchard,* Maryvonne Dendane,* Jean-Franc¸ois Gallard,* Chantal Houe´e-Levin,† Abdelhak Karim,‡ Didier Payen,‡ Jean-Marie Launay,‡ and Claire Ducrocq*,1 *Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, F-91198 Gif sur Yvette, France; †LPCR, Universite´ Paris-sud, F-91405 Orsay, France; and ‡Hoˆpital Universitaire Lariboisie`re 2, rue Ambroise Pare´, F-75010 Paris, France
Received March 10, 1997, and in revised form June 25, 1997
Nitric oxide synthesized in biological systems from Because NO is not very reactive in an oxygen-free buffer, a significant part of serotonin (5-HT) is transformed by NO in nondeaerated phosphate buffer, at pH 7.4, into (4-serotonyl)-4-serotonin, 4-nitrososerotonin, and 4-nitroserotonin. Dimerization and above all nitrosation occur through the HNO2 reaction in the pH 4–6 range, possibly via radical mechanism involving N2O3 . 5-HT is readily a substrate for nitrosation by HNO2 or N2O3 , whereas tyrosine was described as not very reactive under the same conditions. Peroxynitrite converts 5-HT to the (4-serotonyl)-4-serotonin and to the 4-nitro derivative. In order to evaluate whether such structural modifications could modulate the biological properties of 5-HT, arterial pressure was measured after iv bolus injection of these derivatives to anesthetized rats. Injections of the 4-nitroso- and 4-nitro-5-HT resulted in first a brief hypotensive response and did not give the subsequent hypertensive and hypotensive phases observed with 5-HT. Finally, when tested on some cloned rat 5-HT receptors stably transfected into LMTK0 cells, both 4-nitroso and 4-nitro derivatives behaved as agonists and antagonists toward 5HT1B and 5-HT2B receptors, respectively. q 1997 Academic Press
1
To whom correspondence should be addressed. Fax: 33 1 69077247.
L-arginine and oxygen by NO synthases plays crucial
roles in the regulation of a range of physiological functions, such as vasodilation, neurotransmission, and cellular immunity (1). The most reactive targets for NO appear to be the reduced transition metals of proteins such as guanylate cyclase or hemoglobin, oxygen, and the superoxide anion. These reactions account for the short half-life of NO of a few seconds in vivo (2, 3). Current interest is focused on the ability of higher oxides of NO, such as N2O3 or peroxynitrite (ONOO0), to modify peptides, proteins, and other signaling molecules. These nitrogen oxides are short-lived intermediates that are more reactive than their precursor. They are formed during the course of NO oxidation to stable nitrite or during its coupling reaction with superoxide to form nitrate (4, 5). NO-derived nitrogen oxides can also nitrosylate thiols such as cysteine, or cysteinyl residues (6, 7), and oxidize ascorbate and tocopherol. Stimulated macrophages, neutrophiles, and endothelial cells have been shown to generate NO as well as superoxide anion and probably the end-derivative, peroxynitrite. It has been suggested that peroxynitrite is formed in human atherosclerosis and in chronic inflammation (8, 9). Peroxynitrite oxidizes glutathione, ascorbate, and tocopherol and nitrates tyrosine residues in proteins (5).
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We have studied the interactions of NO-related higher oxides under aerobic conditions with serotonin (5-hydroxytryptamine or 5-HT).2 This monoamine hormone plays an important role in the central and peripheral nervous system (10), particularly in regulating blood pressure and organ blood flow (11, 12). The products of the metabolism of 5-HT are of interest not only because of their possible physiological importance but also due to their pathological potentialities. Indeed, the 5-HT levels in the Alzheimer or Parkinsonian brain have been found to be significantly lower than those in age-matched controls, apparently caused by excessive oxidation of 5HT into several unidentified forms (13). A 5-HT dimer has been characterized as the major oxidation product during the respiratory burst of human mononuclear and polymorphonuclear phagocytes (14). In this report, we show that 5-HT is chemically transformed into a dimer and its nitroso and nitro derivatives by nitrogen oxides derived from NO. Particular attention has been paid to the reactivity with nitrous acid because it is the ultimate stable oxidative species derived from NO, and could perhaps be generated in acidic cell compartments (vesicles, hypoxic cells). To assess the proposed mechanism we show the ability of NO2 radical to oxidize 5-HT into its dimer as does azide radical in parallel experiments. In order to determine whether vascular NO might be directly involved in the change of vascular tone via chemical modification of 5-HT, some vascular properties of modified serotonin were investigated. 4-Nitroso and 4-nitro-5-HT administered to anesthetized rats were found to have completely different effects on arterial pressure and cardiac frequency compared to the parent 5-HT. Finally the properties of these derivatives were tested on cloned rat 5-HT receptors stably transfected into LMTK0 cells. EXPERIMENTAL PROCEDURES
Materials. 5-HT hydrochloride was purchased from Sigma, forskolin was from Calbiochem (San 2 Abbreviations used: 5-HT, 5-hydroxytryptamine; DOI, 2,5-dimethoxy-4-iodophenyl-2-aminopropane; ABTS, 2,2 *-azino-bis(3ethylbenzthiazoline-6-sulfonic acid); IP, inositol phosphates; GTI, 5-hydroxytryptamine-O-carboxymethylglycyltyrosinamide; FScA, forskolin-stimulated cAMP accumulation.
Diego, CA); [3H]ketanserin (63.3 Ci/mmol; 1 Ci Å 37 GBq), [3H]5-HT (75 Ci/mmol), and [125I]2,5-dimethoxy-4-iodophenyl-2-aminopropane ([125I]DOI, 2200 Ci/mmol) were from New England Nuclear Research Products (Boston, MA); [125I]5-hydroxytryptamine-5O-carboxymethylglycyltyrosinamide ([125I]GTI, 1860 Ci/mmol) was from Research Biochemicals (Natick, MA). All tissue culture reagents and Hanks’ balanced salt solution were purchased from GIBCO (Grand Island, NY); all other drugs and chemicals (reagent grade) were from Sigma Chemical. Potassium phosphate and sodium azide used in g-irradiation experiments were purchased from Prolabo and sodium nitrite was from Fluka. HPLC system. Aliquots of incubation medium were analyzed with a Waters HPLC system equipped with a Berthold integrator and using a Shandon (C 18 Si, 5 mm) Hypersyl column or a Waters RCM 25 1 10 compact preparative cartridge module. Columns were eluted using a gradient of 5– 45% acetonitrile with 0.05% TFA for 50 min at a flow rate of 1 or 6 ml/min. Products were spectrophotometrically detected by measuring UV absorbance at 215 nm. Molecular mass determination. This was performed on a VG Platform mass spectrometer (VG Biotech, Manchester, UK) using an electrospray ion source and a quadrupole mass analyzer with an upper mass limit of m/z 3000, calibrated using a mixture of polyethylene glycol 300 and 600. Ten-microliter samples were introduced into the source at a flow rate of 5 ml/min (carrier solvent: 50% acetonitrile, 49% water, 1% formic acid); 4000 V was applied to the capillary and 450 V was applied to the counter electrode. The sampling cone voltage was adjusted to obtain the best sensitivity and the lowest level of fragmentation. Experiments were carried out by J.-P. Le Caer (Ecole de Physique et Chimie Industrielles de la Ville de Paris). Reactions with NO. A saturated NO solution was prepared by bubbling NO gas (Air Liquide, France), that had been passed through a deaerated concentrated sodium hydroxide solution (10 molrdm03), into a deaerated phosphate buffer (0.4 molrdm03, pH 7.4), until the concentration of dissolved NO reached 0.7–1 mmolrdm03. The concentration was measured by the amount of nitrite present in oxy-
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genated aliquots measured spectrophotometrically by the Griess reaction (4), or by the absorbance of the oxidized derivative of the 2,2*-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Aliquots of NO solution were added to a 50 mmolrdm03 ABTS solution and NO concentration was measured as a function of the 660 nm and 750 nm absorbances (e Å 12,000 and 15,000 mol01rdm3rcm01, respectively). The NO-saturated solution (900 ml) was added by syringe to 100 ml of a stirred, phosphatebuffered solution of 5-HT (10 mmolrdm03). When NO gas was passed through a solution of sodium hydroxide and then gently bubbled into a solution of 5-HT (20 mmolrdm03) in nondeaerated phosphate buffer (0.4 molrdm03, pH 7.4) for 2 min at room temperature, HPLC analysis indicated that 5-HT was entirely converted into the same compounds as those obtained with HNO2 . Their identities were confirmed by electrospray MS analysis. 5,5*-Dihydroxy-4,4*-bitryptamine 2 and 4-nitroso5-hydroxytryptamine 3. Two or 5 eq of sodium nitrite was added to a solution of 10 or 40 mmolrdm03 5-HT hydrochloride in 0.2 molrdm03 acetate buffer adjusted to pH 5. The mixture, stirred a few minutes, became purplish red and was left at room temperature. The reaction was monitored by HPLC. The dimer (12 min retention time under conditions described above) and the 4-nitroso 5-HT (28 min) derivatives were produced after 4 h of incubation and were recovered by preparative HPLC and then lyophilized. 5,5*-Dihydroxy-4,4*-bitryptamine 2 analysis. Electrospray MS: m/z 351; 1H NMR 400 MHz in D2O: CH2 –CH2 (4m, 2.3, 2.5, 2.6, and 2.7 showing nonequivalent H); H-2 (s, 7.3), H-6 (d, 7.1, J6 – 7 Å 9 Hz), H-7 (d, 7.6, J6 – 7 Å 9 Hz) slightly different from the spectrum obtained in DMSOd6 (15); lmax Å 310 nm, in water at pH 2–3. Centesimal analysis showed that the dimer was a trifluoroacetate salt containing 2.5 water molecules, Mr 678. Nitroso derivative 3 analysis. Electrospray MS: m/z 206 and 189 (MH/ 0 NH2); 1H NMR 400 MHz in D2O: CH2 –CH2 (4 m, 2.5, 2.65, 2.75, and 2.85 at 274K which collapsed to 3 signals at 2.9, 3.1, and 3.2 at 300K). Loss of the signal from C4 –H observed in the spectrum of 1 is accompanied by the disappearance of long range coupling between C4 –H and
C6 –H. Each aromatic proton gave two peaks: H-6 (2d, 4.9 and 5.05, J6 – 7 Å 9 Hz), H-2 (2s, 6.55, 6.65), H-7 (2d, 6.9, 7.0, J6 – 7 Å 9 Hz). The intensities of the 4.9, 6.55, and 6.9 signals were twice those observed at 274K and almost equal to those observed at 300K. The spectrum of 3 in acidic water indicated lmax Å 520 nm (e Å 3700 mol01rdm3rcm01) and 385 nm (e Å 3400 mol01rdm3rcm01). Centesimal analysis showed that product 3 of Mr 355 was a trifluoroacetate salt containing 2 water molecules. 4-Nitro-5-hydroxytryptamine 4. Peroxynitrite was synthesized and its concentration was determined spectroscopically, according to Beckman’s method (16). It was added to 1 ml of a stirred solution of 5-HT (1 mmolrdm03) in phosphate buffer (0.4 molrdm03, pH 7.4) at 257C. HPLC analysis was performed on a 50-ml aliquot. The potential effects of nitrite, nitrate, and H2O2 , all present in peroxynitrite, were tested allowing the latter to decompose in the phosphate buffer before the addition of 5-HT. Less than 2% of the dimer 2 formed by the peroxynitrite reaction, was produced by residual H2O2 . The prepared nitro derivative 4, purified by HPLC, lyophilized, and dried (10% yield using 3 HOONO per 5-HT) was characterized by a 420-nm absorbance in water, pH 3. The electrospray MS spectrum showed a peak at m/z 222. 1H NMR 400-MHz spectra performed in D2O showed the CH2 (t, 3.2, 4H) and the aromatic protons H-2 (s, 7.5), H-6 (d, 6.9, J Å 9 Hz) and H-7 (d, 7.7, J Å 9 Hz). The loss of the C4–H signals and the disappearance of long-range coupling between C4–H and C6–H compared to the spectrum of 1 showed that the nitro group substitution had occurred at the 4-C position. Centesimal analysis showed that it was a trifluoroacetate salt of 4-nitro 5-HT 4, Mr 392. Nitrosation kinetics of 5-HT. These were obtained by following 4-nitroso-5-HT 3 formation at 385 nm. All experiments were carried out at room temperature, using a 941 Kontron UV-visible spectrometer. Reaction with NO2 . g-radiolysis experiments were performed with a 137Cs irradiator (Cis-Bio) located at Institut Curie-section de Recherche (Orsay, France). The dosimetry was performed using Fricke’s procedure (17). Before irradiation, solutions of sodium nitrite (1002 molrdm03) or sodium azide
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(1002 molrdm03) in potassium phosphate buffer (10 mmolrdm03, pH 7.4) were equilibrated with N2O by flushing at least 60 min under permanent vortexing, thus avoiding bubbling in the solution. The NO2 radicals were created by scavenging OH radicals by nitrite ions under N2O (18), following the reaction sequence:
a transducer system (Propac; Abbott Lab, Chicago, IL). The transducer was connected to an amplifier (CGR Thomson, Les Ulis, France) linked to a digital/ analog converter (BIOPAC Mac Pac System) and a microcomputer. After a stabilization period, 5-HT, 4-nitroso-5-HT 3, or 4-nitro-5-HT 4 diluted in a constant volume of sterile water was injected into the right jugular vein. Injections were performed in random order and a 1-h delay separated each injection. Such continuous recording of arterial pressure allowed verification of the time-related peak variations in pressure and heart rate. Receptors binding assays and measurement of 5HT1B and 5-HT2B receptor responses. Stable transfections of LMTK0 cells with the rat 5-HT1B , 5-HT2A , 5-HT2B , and 5-HT3 genes, cultures of these cell lines, membrane preparation, receptor binding assays, and measurements of receptor responses, i.e., inositol phosphates (IP) accumulation (5-HT2A , 5-HT2B) or inhibition of forskolin-stimulated cAMP accumulation (FScA) (5-HT1B), were performed as described previously (19, 20).
0 / N2O r •OH / HO0 / N2 eaq •
OH / NO20 r HO0 / •NO2
The N3 radicals were created by scavenging the OH radical formed by irradiation of azide-containing solutions under a N2O atmosphere according to the following reaction: •
OH / N30 r HO0 / •N3
The yields of NO2 or of N3 radicals are thus both equal to GOH / Ge-aq , i.e., to 0.55 mmolrJ01. At a dose rate of 3.89 Gyrmin01, the NO2 radicals were created at a rate of 2.14 mmolrdm03rmin01. Hemodynamic responses in anesthetized rats. After ip injection of 50 mgrkg01 pentobarbital, the anesthetized males CD rats (380–400 g) were mechanically ventilated via tracheotomy (3 ml tidal volume, 65–70 breath rate/min). The adequacy of the ventilation was checked by serial blood gas measurements. The arterial pressure was continuously monitored in the carotid vessel after catheterisation using
H
RESULTS
When 5-HT 1 was treated at room temperature with a solution of NO in an aerobic buffered solution at pH 7.4, or by using nitrite at pH 5, it was essentially converted, in both cases, into a mixture of the symmetrical dimer 5,5*-dihydroxy-4,4*-bitryptamine 2 and of the 4-nitroso derivative 3.
O N
N
NO¤ NH¤
HO NH¤
OH HO
NH¤
HO
NH¤
N
N
N
H
H
H 2
4
3
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treated with 5 eq of nitrous acid at pH 6 was nitrosated at a rate of 3 1 1005 molrdm03rmin01, that is, tenfold slower than at pH 5 and a thousandfold slower than at pH 3. Independently of pH, the nitrosation rate was a function of the 5-HT and nitrite concentrations. The nitrosation rate was proportional to the initial nitrite concentration when the nitrite/ 5-HT ratio was higher than two (results not shown) and otherwise varied with the square of the 5-HT concentration (Fig. 2). The reaction rate was second order with respect to the concentration of 5-HT due to a complex effect of the pH on this reaction, in a similar way to 2-naphthol nitrosation (21). FIG. 1. Kinetics of dimer (h) 2 and 4-nitroso-5-HT (m) 3 formation from 5-HT (1 mmolrdm03) and nitrite (2 mmolrdm03) in acetate buffer at pH 5. Under these conditions, 5-HT decreased linearly in the 2 h first time. Concentrations were evaluated by calibrating HPLC integration with known concentrations of each compound.
The structures of 2 and 3 were established by MS and NMR studies. The 1H NMR spectrum of 3 showed the presence of two distinct stable conformers of 4-nitroso 5-HT. These structures result from a hydrogen bond between the nitroso group and the primary amine, corresponding to a fixed side chain, or a hydrogen bond involving the aromatic hydroxyl group. Increasing the temperature promoted formation of the latter conformer. Reactions of 5-HT with NO. There was a partial transformation with a single bolus of NO injected into a neutral solution of 5-HT (NO/5-HT Å 1/1 mmolrdm03), leading to 8% nitrosation and 2% dimerization. Because NO was unreactive in oxygenfree buffer, molecular oxygen is needed for NO reactivity: nitrosating agents, such as NO2 or N2O3 , may be involved in these reactions. Nitrogen oxide intermediates of NO oxidation were either rapidly transformed into nitrite or irreversibly trapped by the substrate. Furthermore, NO behaved also as a nitrating agent in the presence of oxygen as pointed out by the formation of a small quantity of 4-nitro5-HT 4 (1.2%), which was clearly not detectable in the HNO2 reaction. Reactions of 5-HT with HNO2 . The reactions with nitrous acid accounted for the pH-dependent total conversion of 5-HT to both the nitroso derivative 3 and the dimer 2 (Fig. 1). Thus, 5-HT (20 mmolrdm03)
Reactions of 5-HT with peroxynitrous acid. 5-HT was converted by peroxynitrite in aqueous buffered solutions at pH 7.4 into the nitrocompound 4 and the dimer 2. The best peroxynitrite to 5-HT ratio for obtaining the dimer was 2/1, while it was 7/1 for the nitro compound (Fig. 3). The products formed from 5-HT treated with higher excess of peroxynitrite were not detected under our conditions. We evaluated the effects of some natural antioxidants on the yields of dimerization and nitration with peroxynitrite (Fig. 4). Ascorbate and cysteine decreased the yields of products in all the reactions involved, providing 100% inhibition with an equimolar quantity of ascorbate (compared to 5-HT concentration) and 50% inhibition with cysteine. Conversely, ferric ions significantly increased (20%) the
FIG. 2. Influence of the initial 5-HT concentration on the nitrosation rate of 5-HT by nitrite (5 eq) in acetate buffer at pH 5. Nitrosation rates were evaluated by spectrophotometry using the characteristic 385-nm absorbance of the 4-nitroso-5-HT 3.
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FIG. 3. Yields of the dimer (h) 2 and the nitro derivative (j) 4 from the reaction of 5-HT (1 mmolrdm03 in phosphate buffer at pH 7.4) with peroxynitrite. 5-HT was converted linearly with peroxynitrite concentration until 3 eq. Concentrations were evaluated by calibrating HPLC integration with known concentrations of each compound.
nitration yield without affecting that of dimerization. Reaction of 5-HT with •NO2 . Table I gives the yields of compounds obtained by reaction of 5-HT with either •NO2 or azide free radicals in bufferedphosphate solutions. The amount of dimer 2 produced is almost the same for both oxidants, showing a very similar oxidation proceeding by a simple outer-sphere mechanism. As observed for tyrosine, only a part of the free radicals undergoes dimerization during the oxidation process (22). The kinetic scheme proposed is:
447
ing moderate hypotention (023%) occurring at 6 { 2 s followed by a large and significant increase in pressure (/59 { 8%; õ0.001) peaking at the 25th second after bolus injection. This hypertension was followed by a stable hypotension (037 { 6%; õ0.01). The heart rate had a different evolution: after a significant bradycardia (042.4 { 23%; õ0.001) occurring at the 6th second, the heart rate normalized thereafter. In terms of pressure response, bolus injections of the 4nitroso or 4-nitro derivatives induced initial hypotension similar to 5-HT but with no delayed hypertension (õ0.001) and a more rapid and complete pressure recovery (Fig. 6). In addition, the initial bradycardia induced by 5-HT did not occur when 5-HT was substituted on the C4 position (Fig. 7). Receptors binding assays and measurement of 5HT1B and 5-HT2B receptor responses. Since the 5HT triphasic hemodynamic pattern is attributed to the successive activation of 5-HT3-, 5-HT2-, and 5HT1-like receptors (12), the 4-substituted derivatives of 5-HT were tested on stably transfected cells with subtypes of these 5-HT receptors present in rat vasculature (23). Table II shows that both 4-nitroso-5HT 3 and 4-nitro-5-HT 4 are able to interact significantly with rat 5-HT1B and 5-HT2B receptors but not with rat 5-HT2A or 5-HT3 receptors. Both derivatives exhibit similar binding affinities which, compared to 5-HT itself, are lower for the 5-HT1B receptor and alike for the 5-HT2B one. Interestingly enough, 3 and 4 inhibit FScA (5-HT1B receptor) or DOI-induced IP
5-HT / •N3 or •NO2 r •5-HT / N30 or NO20 [a] 2(•5-HT) r 2
[b]
5-HT / NO2 r 4
[c]
•
•
Dimerization [b] should compete with the nitration [c] in the presence of •NO2 . Hemodynamic responses in anesthetized rats. Before each injection, arterial pressure did not differ from baseline. After an iv bolus injection of 5-HT, three successive phases were observed: a short-last-
FIG. 4. Influence of some factors on the yields of dimerization and nitration of 5-HT (1 mmolrdm03) by peroxynitrite (2 mmolrdm03). Concentrations were evaluated by calibrating HPLC integration with known concentrations of each compound.
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BLANCHARD ET AL. TABLE I
Yields of 5-HT Derivatives after g-Irradiation of Neutral Nitrite or Azide Solutions •
•
NO2
N3
Irradiation dose (Gy)
19.4
38.9
58.35
77.8
116.7
155.6
5-HT dimer 2 (%) Residual 5-HT (%) 4-Nitro-5-HT 4 (%)
8{1 83 { 4 10 { 1
8 {2 81 { 4 10.5 { 1
7 {1 68.5 { 2 12 { 1
8{1 64 { 3 13 { 1
8.5 { 2 54 { 2 14 { 1
13 { 2 62 { 2 0
Note. The NO2 or N3 radicals were created by scavenging OH by nitrite (1002 mol rdm03) or azide (1002 molrdm03) in phosphate buffer (1002 molrdm03, pH 7.4) under N2O. The initial concentration of 5-HT was 1004 molrdm03. Concentrations of the 5-HT derivatives were evaluated by calibrating HPLC integration with known concentrations of each compound.
accumulation (5-HT2B receptor), indicating that these derivatives behave as agonists and antagonists toward the rat 5-HT1B and 5-HT2B receptors, respectively.
There are both ionic and radical routes which could lead to formation of dimer 2 and nitroso derivative 3. Activation of the C4 position toward nucleophilic attack results from a carbocation intermediate which should give 4,5-dihydroxytryptamine in water and thus the corresponding 4,5-dione (26). A radical mechanism could be also proposed to account for both dimerization and nitrosation of 5-HT by N2O3 (27). It has been suggested that NO2 can capture a hydrogen atom from the hydroxyl group of 5-HT forming a phenoxy radical (28) which can be delocalized on the aromatic ring. This reaction occurs between 5-HT and NO2 obtained by g-irradiation of nitrite ions under N2O. In the absence of NO, dimerization of 5-HT occurs with the same rate and with the same yield whether with azide or NO2 radicals (Table I). The mechanism is close to that of one electron oxidation of tyrosine in aqueous solution and in proteins which leads to dityrosine and also to hydroxylated compounds (22, 29). The radiolytic yield of dityrosine formation using azide as oxidant is
DISCUSSION
The aim of our work was to study the reactivity of 5-HT with the oxidized derivatives of the NO radical, these entities being able to occur in mammals. Nitrous acid, the end-product of NO autoxidation as well as NO in the presence of oxygen at neutral pH, gave 4-nitroso-5-HT 3 and 5,5*-dihydroxy-4,4*-bitryptamine 2 in similar proportions (4/1, respectively). These results suggest that the same reagent, N2O3 (or the •NO2 and •NO couple), may be formed either by autoxidation of NO or through HNO2 dismutation (24, 25). All NO derivatives are oxidizing agents since they all led to dimerization of 5-HT, the major reaction product of 5-HT oxidation, as determined conclusively by electrochemical methods (15).
TABLE II
Affinity Values of 5-HT, 4-Nitroso-5-HT, and 4-Nitro-5-HT for Cloned Rat 5-HT Receptors Stably Transfected into LMTK0 Cells
5-HT 4-Nitroso-5-HT 3 4-Nitro-5-HT 4
5-HT1B
5-HT2A
5-HT2B
5-HT3
7.60 { 0.13 6.97 { 0.15 6.83 { 0.22
5.51 { 0.17 õ5 õ5
7.59 { 0.15 7.66 { 0.17 7.31 { 0.18
6.89 { 0.08 õ5 õ5
Note. Affinity values (pKD Å 0log molrdm03) were determined using the following radioligands: [125I]GTI for 5-HT1B , [3H]ketanserin for 5-HT2A , [125I]DOI for 5-HT2B , and [3H]5-HT for 5-HT3 . Nonspecific bindings were determined in the presence of 10 mM mianserin (5-HT2 sites) or 10 mM 5-HT (5-HT1B and 5-HT3 sites). Each value is the mean { SE of at least three independent trial runs in triplicate. Eight different concentrations of each competing drug were used. Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.
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449
FIG. 5. Proposed scheme for the reactivity of nitrous acid at pH 5 or NO in the presence of oxygen at pH 7.4.
around 0.1 mmol J01 at pH 7 (30), lower that of the dimer obtained from 5-HT. The dimerization is more rapid than the formation of the nitro derivative 4 with NO2 . In the presence of NO, the serotonyl radical might react more rapidly with NO than with NO2 , asserting the proposed mechanism of nitrosation and oxidation of 5-HT with HNO2 or N2O3 (Fig. 5). Nitrogen oxides derived from NO under aerobic conditions are mainly nitrosating agents and to a lesser extent nitrating agents, suggesting that NO2 is an intermediate formed in addition to N2O3 during NO autoxidation (31). In addition to oxidation, nitration is an essential reaction of 5-HT in the presence of peroxynitrite. The yield of peroxynitrite-mediated nitration increased in the presence of ferric ions, indicating an ionic mechanism involving NO2/ from peroxynitrous acid HOONO (5, 32). Dimerization and perhaps a part of nitration by peroxynitrite could also take place via a radical mechanism, in which peroxynitrous acid undergoes homolytic rupture with the subsequent formation of •OH and •NO2 . Both radi-
cals can generate a serotonyl radical from the substrate. However, the •OH-generating system (Fe2/, ascorbate, H2O2) is known to oxidize 5-HT to compounds that we have not detected: 5,6-dihydroxytryptamine, 5-hydroxy-3-ethylamino-2-oxindole, and a dimer formed by linkage between 7-(tryptamine-4,5dione)-yle and 3-(5-hydroxy-tryptamine-2-one)-yle (33, 34). The biological properties of the proposed new metabolites 3 and 4 were evaluated on arterial pressure in rats. The triphasic vascular response characteristic of 5-HT consists of an initial quick fall in arterial pressure followed by a pressor response of 40 s and finally a long-lasting depressor phase (Fig. 6). This hemodynamic pattern is admittedly related to the successive activation of 5-HT3 , 5-HT2 , and 5-HT1 receptors (12). Administration of the 4-nitroso 3 or 4nitro 4 derivatives gave the same hypotensive responses as 5-HT, corresponding to 5-HT3 and 5-HT1 agonist properties (Fig. 6). The hypertension peak (Fig. 6) observed after 5-HT administration was clearly suppressed in the case of both 4-substituted derivatives. Accordingly, both derivatives behave as
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sponses similar to those of 5-HT (Fig. 6). The longlasting depressor phase fits well with their 5-HT1B agonist properties (Table I), but the rapid initial drop of arterial pressure, attributed to 5-HT3 receptors (12), appears at variance with the apparent nonrecognition of this 5-HT receptor type by both derivatives 3 and 4 (Table I). This might be due to the cloning and expression of the only ligand binding subunit of the 5-HT3 receptor, therefore not reflecting the heterogeneity observed with this receptor, not only between but also within species (35). This might also be due to involvement of other 5-HT receptors with similar antagonist pharmacophores, such as 5-HT4 (35). This latter possibility is favored by the inability of both derivatives 3 and 4 to reproduce the 5-HT-induced bradycardia (the so-called Bezold–Jarisch reflex, Fig. 7), also attributed to 5HT3 receptors despite the multiplicity of 5-HT receptors in the heart (36). Clearly, the delineation of the pharmacological profiles of derivatives 3 and 4 deserves further investigations. CONCLUSION
The conversions of 5-HT we have observed are very similar to those described for tyrosine (32), even
FIG. 6. Typical recordings of the arterial pressure response provoked by iv bolus of 5-HT or 4-nitroso-5-HT. Mean arterial pressure obtained after 50 mg iv injection of 5-HT (black column), n Å 6, 4-nitroso-5-HT (open column), n Å 8, or 4-nitro-5HT (hatched column), n Å 5, in anesthetized rats. The arterial pressure was evaluated at time corresponding to each phase of the vascular response following 5-HT administration. Means / SE from n experiments.
antagonists toward rat 5-HT2B receptors, suggesting that this 5-HT2 subtype, and not the 5-HT2A receptor (Table I), accounts, at least partly, for these suppressive effects. Administration of the 4-nitroso 3 or 4-nitro 4 derivatives resulted in hypotensive re-
FIG. 7. Heart rate (%) in each phase following iv bolus doses of 50 mg of 5-HT (black column), 4-nitroso-5-HT (open column), or 4-nitro-5-HT (hatched column) in anesthetized rats. The mean heart rate was evaluated at time corresponding to each phase of the vascular response following 5-HT administration.
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though 5-HT is more sensitive to oxidation than tyrosine. Indeed, 5-HT has been described as a true radical scavenger of oxygen and its reduced derivatives. It also behaves as a scavenger of oxidizing and nitrating agents derived from NO. The concentration of free 5-HT in the plasma is in the picomolar range. 5-HT can be released in large amounts from its storage centers in platelets or thrombocytes and plays a protective role at sites of endothelial damage during the course of inflammation or infection. Some authors have pointed out its ability to suppress the respiratory burst and phagocytic activity and to protect endothelial cells at inflammation sites, via the dimerization of 5-HT and subsequent reduction of the reactive oxygen species (14). On the other hand, phagocytic function should be preserved because phagocytes can actively take up and store 5-HT in granules. In any interpretation of the various effects of modulation of the immune system by 5-HT, direct interactions between reactive agents derived from O2 or/and NO should be seriously considered. Numerous biological interactions between NO and 5-HT are postulated to involve recognition of 5-HT receptors and further metabolic pathways, among which are activation of NO synthase (37, 38). In the nervous and peripheral systems, the activity of 5HT (vasopressure and intestinal motility, mood and behavior) is known to be linked to the capacity of this amine to interact with its specific receptors. Chemical interactions between NO and 5-HT should modulate the biological actions of 5-HT in NO-synthase-containing cells or in tissues (39). Particular interest should be focused on behavioral abnormalities displayed in male mice lacking neuronal nitric oxide synthase, similar to those obtained with targeted disruption of 5-HT1B receptor genes or with drugs inducing 5-HT depletion (40). The detection of such irreversibly modified derivatives of 5-HT in cells or in tissues is under investigation. ACKNOWLEDGMENTS We thank Dr. Yann Henry for his valuable advice and Dr. Pierre Potier for encouragement. Financial support was from the Centre National de la Recherche Scientifique and Ministe`re de la Recherche et de la Technologie (France).
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